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      Full-length RNA-seq from single cells using Smart-seq2.

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          Abstract

          Emerging methods for the accurate quantification of gene expression in individual cells hold promise for revealing the extent, function and origins of cell-to-cell variability. Different high-throughput methods for single-cell RNA-seq have been introduced that vary in coverage, sensitivity and multiplexing ability. We recently introduced Smart-seq for transcriptome analysis from single cells, and we subsequently optimized the method for improved sensitivity, accuracy and full-length coverage across transcripts. Here we present a detailed protocol for Smart-seq2 that allows the generation of full-length cDNA and sequencing libraries by using standard reagents. The entire protocol takes ∼2 d from cell picking to having a final library ready for sequencing; sequencing will require an additional 1-3 d depending on the strategy and sequencer. The current limitations are the lack of strand specificity and the inability to detect nonpolyadenylated (polyA(-)) RNA.

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          Author and article information

          Journal
          Nat Protoc
          Nature protocols
          Springer Science and Business Media LLC
          1750-2799
          1750-2799
          Jan 2014
          : 9
          : 1
          Affiliations
          [1 ] Ludwig Institute for Cancer Research, Stockholm, Sweden.
          [2 ] 1] Ludwig Institute for Cancer Research, Stockholm, Sweden. [2] Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
          Article
          nprot.2014.006
          10.1038/nprot.2014.006
          24385147
          4ea48f1d-25a2-4bb8-8bcf-03cbfa096ecb
          History

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